This invention relates to isoelectric focusing, and particularly to methods for mobilizing a focused pattern of ampholytes such as, for instance, proteins in a separation medium for purposes of recovery, detection or both.
High performance liquid chromatography (HPLC) is popular for both analytical and small scale preparative purposes, due to its ability to provide both high speed and high resolution of very small samples. Electrophoresis, including isoelectric focusing, may also be done on a small scale, with similar advantages of speed and resolution.
To achieve speeds similar to those obtainable in HPLC systems, electrophoresis-based systems must use detection techniques which avoid staining and derivatization. The most suitable techniques are light absorption measurements, done either by scanning the medium in which the zones are focused, or by mobilizing the focused zones past a single detection point in the medium itself or out of the medium into a detection cuvette.
Mobilization has the advantage of not requiring motorized scanning equipment. It further enables one to recover the isolated zones individually for preparative purposes. Mobilization by pumping a solution through the tube or other vessel in which the medium is held to purge the vessel by hydrodynamic flow has previously been used following isoelectric focusing in sucrose gradients (without voltage applied). In this case, the parabolic zone distortion caused by the pumping is suppressed by the sucrose gradient, although zone broadening by diffusion still occurs. This way of suppressing zone distortion only works when the column has a relatively large diameter, for instance 3-20 mm, and only when it is mounted in a vertical position. For narrow-bore columns (diameters in the range 0.05-2 mm) sucrose gradients will not suppress the parabolic zone distortion occurring during the pumping procedure. Mobilization of isoelectrically focused zones by pumping cannot, of course, be used when the focusing is performed in a gel.
U.S. patent application Ser. No. 06/787,291, filed Oct. 15, 1985, commonly owned and copending herewith, discloses a mobilization method consisting of changing the pH of either the anolyte or the catholyte or both after the focusing step to provide both anolyte and catholyte with pH's varying in the same direction from the isoelectric points of each of the ampholyte zones focused in the separation medium. The voltage which is then applied across the medium causes the entire protein zone pattern to move as a unit out of the medium. The preferred embodiment in the disclosure involved replacing the anolyte used in the focusing step with the catholyte, or vice versa, with the result that the same solution was in contact with both ends of the separation medium.